In this script we are going to map myeloid cell subtypes onto the Visium slides.
library(Seurat)
library(ggpubr)
library(cowplot)
library(dplyr)
library(ggplot2)
library(stringr)
library(readr)
library(SPOTlight)
Loading necessary paths and parameters
set.seed(123)
source(here::here("misc/paths.R"))
source(here::here("utils/bin.R"))
"{epithelium}/{plt_dir}" %>%
glue::glue() %>%
here::here() %>%
dir.create(path = ,
showWarnings = FALSE,
recursive = TRUE)
"{epithelium}/{robj_dir}" %>%
glue::glue() %>%
here::here() %>%
dir.create(path = ,
showWarnings = FALSE,
recursive = TRUE)
Extract sample id and get Donor ID
# sample_id <- params$sample_id
sample_id <- "esvq52_nluss5"
donor_id <- id_sp_df[id_sp_df$gem_id == sample_id, ] %>% dplyr::pull(donor_id)
We have 4 different datasets that we are going to analyze separately. The spatial data comes from the script 03-clustering/03-clustering_integration.Rmd while the sc data can be found in Ramon’s scRNAseq analysis: /scratch/devel/rmassoni/tonsil_atlas_private/2-DOWNSTREAM_PROCESSING/results/R_objects/processed_seurat_objects/processed_seurat_objects/tonsil_integrated_with_harmony_scrublet_annotated.rds.
sp_obj <- "{clust}/{robj_dir}/integrated_spatial.rds" %>%
glue::glue() %>%
here::here() %>%
readRDS(file = .)
# Single cell data
sc_obj <- "{decon}/{robj_dir}/tonsil_integrated_with_harmony_scrublet_annotated.rds" %>%
glue::glue() %>%
here::here() %>%
readRDS(file = .)
# level 5 Epithelium T Cells
lvl4_epi_obj <- "{epithelium}/{robj_dir}/epithelial_annotated_level_4.rds" %>%
glue::glue() %>%
here::here() %>%
readRDS(file = .)
We are going to use SPOTlight to deconvolute the spatial spots and map the cell types.
We will start by adding the level5 definitive metadata labels to the full object.
lvl4_metadata <- lvl4_epi_obj@meta.data %>%
dplyr::select(barcode, annotation_level_3)
table(lvl4_metadata$annotation_level_3)
##
## Basal cells Crypt FDCSP epithelium Outer surface Surface epithelium VEGFA+
## 55 88 33 14 58 29
sc_obj@meta.data <- sc_obj@meta.data %>%
tibble::rownames_to_column("barcode") %>%
dplyr::left_join(lvl4_metadata, by = "barcode") %>%
dplyr::mutate(
# Annotation for SPOTlight
annotation_deconv = dplyr::if_else(
is.na(annotation_level_3), annotation_level1, annotation_level_3)
# Annotation for SPOTlight with condensed names
# annotation_cons_deconv = dplyr::case_when(
# annotation_deconv %in% c("Central Mem PASK-", "Central Mem PASK+") ~ "Central Mem",
# annotation_deconv %in% c("Follicular Th CXCL13+CBLB+_0", "Follicular Th CXCL13+CBLB+_1", "Follicular Th CXCL13+CBLB+_2") ~ "Follicular Th CXCL13+",
# annotation_deconv %in% c("Central Mem PASK-", "Central Mem PASK+") ~ "Central Mem PASK",
# TRUE ~ annotation_deconv)
) %>%
# Remove CD4 T cells not anotated in Level 5 since they have been removed in the process
dplyr::filter(! annotation_deconv %in% c("epithelial", "unknown")) %>%
tibble::column_to_rownames("barcode")
Subset to 500 cells per cell type
#### Get cell IDs to subset by cluster ####
keep_ids <- lapply(
split(sc_obj@meta.data, sc_obj@meta.data[, "annotation_deconv"]),
function(subdf) {
# Determine n_sample, if the size of the group is < cl_n use all the group, if not just use cl_n
n_sample <- dplyr::if_else(nrow(subdf) < 500,
as.numeric(nrow(subdf)),
as.numeric(500))
# Subset a random selection of that group and get the identifiers
tmp_ds <- subdf[sample(seq_len(nrow(subdf)), n_sample), ] %>%
tibble::rownames_to_column("barcodeID") %>%
dplyr::pull(barcodeID)
return(tmp_ds)
}) %>%
purrr::flatten_chr() # flatten the list into a vector
#### Subset seurat object ####
sc_obj_sub <- sc_obj[, keep_ids]
Since the normalization was carried out with the entire dataset we are going to do normalization again within this subset
sc_obj_sub <- Seurat::NormalizeData(
sc_obj_sub,
normalization.method = "LogNormalize",
scale.factor = 10000,
verbose = FALSE) %>%
Seurat::FindVariableFeatures(., nfeatures = 3000, verbose = FALSE) %>%
Seurat::ScaleData(., verbose = FALSE)
1st we need to determine the marker genes for each high-level cluster
markers_lvl1_out <- "{epithelium}/{robj_dir}/markers_lvl1_epith.RDS" %>%
glue::glue() %>%
here::here()
if (file.exists(markers_lvl1_out)) {
markers_lvl1 <- readRDS(file = markers_lvl1_out)
} else {
#### Extract marker genes from each cluster ####
Seurat::Idents(object = sc_obj_sub) <- sc_obj_sub@meta.data[, "annotation_level1"]
markers_lvl1 <- Seurat::FindAllMarkers(
object = sc_obj_sub,
assay = "RNA",
slot = "data",
verbose = TRUE,
only.pos = TRUE,
max.cells.per.ident = 200)
saveRDS(object = markers_lvl1, file = markers_lvl1_out)
}
DT::datatable(markers_lvl1, filter = "top")
Now find the markers for CD4 T cells level 5 only between CD4 T cells
markers_lvl4_out <- "{epithelium}/{robj_dir}/markers_lvl4_epithelial.RDS" %>%
glue::glue() %>%
here::here()
if (file.exists(markers_lvl4_out)) {
markers_lvl4 <- readRDS(file = markers_lvl4_out)
} else {
# lvl4 myeloid cells lvl4_metadata
lvl4_subs <- subset(
sc_obj_sub,
subset = annotation_deconv %in% unique(lvl4_metadata$annotation_level_3))
#### Extract marker genes from each cluster ####
Seurat::Idents(object = lvl4_subs) <- lvl4_subs@meta.data[, "annotation_deconv"]
markers_lvl4 <- Seurat::FindAllMarkers(
object = lvl4_subs,
assay = "RNA",
slot = "data",
verbose = TRUE,
only.pos = TRUE,
max.cells.per.ident = 200)
saveRDS(object = markers_lvl4, file = markers_lvl4_out)
}
DT::datatable(markers_lvl4, filter = "top")
Join markers from both runs. Since we still want to keep the epithelial cells markers in the lower level markers we will add them to each one.
# Keep markers for major cell types
# gene_vec <- c("BCL6", "PRDM1", "FDCSP")
markers_lvl1_sub <- markers_lvl1 %>% dplyr::filter(cluster != "epithelial") %>%
dplyr::filter( ( avg_log2FC > 1 | pct.1 - pct.2 > 0.75) &
p_val_adj < 0.01 & pct.1 > 0.3)
# | gene %in% gene_vec)
# Keep markers for general epithelial T cells
markers_lvl1_epith <- markers_lvl1 %>%
dplyr::filter(cluster == "epithelial") %>%
# Filter to get CD4 specific markers
# We are keeping those genes with a logFC > 1 or a pct.1 - pct.2 > 0.75 which
# have a p_val_adj < 0.01 & pct.1 > 0.6 while keeping CD4
dplyr::filter( ( avg_log2FC > 1 | pct.1 - pct.2 > 0.75) &
p_val_adj < 0.01 & pct.1 > 0.6)
Add general Epithelial cell markers to the specific cell types
markers_lvl4_update <- lapply(as.character(unique(markers_lvl4$cluster)), function (i) {
# Filter level 5 clusters
tmp <- markers_lvl4 %>%
# dplyr::filter(avg_log2FC > 0.75 | pct.1 - pct.2 > 0.4) %>%
# dplyr::filter(avg_log2FC > 0.5 | pct.1 - pct.2 > 0.4) %>%
dplyr::filter(cluster == i) %>%
# Remove ribosomal genes
dplyr::filter(!stringr::str_detect(string = gene,
pattern = "^RPS|^RPL"))
# Add general CD4 markers
# tmp %>%
# dplyr::bind_rows(markers_lvl1_epith) %>%
# dplyr::mutate(cluster = i)
}) %>%
dplyr::bind_rows()
DT::datatable(markers_lvl4_update, filter = "top")
Combine level-1 and level-4 markers
all_markers <- dplyr::bind_rows(markers_lvl1_sub, markers_lvl4_update) %>%
dplyr::arrange(dplyr::desc(avg_log2FC)) %>%
dplyr::group_by(cluster, gene) %>%
dplyr::filter(dplyr::row_number() == 1)
DT::datatable(all_markers, filter = "top")
2nd run deconvolution
Seurat::Idents(object = sc_obj_sub) <- as.character(sc_obj_sub@meta.data$annotation_deconv)
spotlight_ls <- spotlight_deconvolution(
se_sc = sc_obj_sub,
counts_spatial = sp_obj@assays$Spatial@counts,
clust_vr = "annotation_deconv",
cluster_markers = all_markers,
cl_n = 100,
hvg = 0,
ntop = NULL,
transf = "uv",
method = "nsNMF",
min_cont = 0)
## [1] "Preparing Gene set"
## [1] "Normalizing count matrix"
## [1] "Seeding initial matrices"
## [1] "Training..."
## [1] "Time to train NMF model was 16.48mins"
## [1] "Deconvoluting spots"
##
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decon_fn <- "{epithelium}/{robj_dir}/spotlight_ls_epithelium.rds" %>%
glue::glue() %>%
here::here()
# if (! file.exists(decon_fn) ) {
saveRDS(
object = spotlight_ls,
file = decon_fn)
# } else {
# spotlight_ls <- readRDS(file = here::here(glue::glue("{epithelium}/{robj_dir}/spotlight_ls_cd4.rds")))
# }
3rd Check Topic profiles
nmf_mod_ls <- spotlight_ls[[1]]
nmf_mod <- nmf_mod_ls[[1]]
h <- NMF::coef(nmf_mod)
rownames(h) <- paste("Topic", 1:nrow(h), sep = "_")
topic_profile_plts <- SPOTlight::dot_plot_profiles_fun(
h = h,
train_cell_clust = nmf_mod_ls[[2]])
topic_profile_plts[[2]] +
ggplot2::theme(axis.text.x = ggplot2::element_text(angle = 90),
axis.text = ggplot2::element_text(size = 12))
decon_mtrx <- spotlight_ls[[2]]
decon_mtrx <- decon_mtrx[, colnames(decon_mtrx) != "res_ss"]
# Set as 0 cell types predicted to be under 1 % of the spot
# decon_mtrx[decon_mtrx < 0.03] <- 0
Change column names
nm_df <- data.frame(
mod_nm = stringr::str_replace_all(string = unique(all_markers$cluster),
pattern = "[[:punct:]]|\\s|\\+" ,
replacement = "."),
plt_nm = unique(all_markers$cluster)
)
# Save nm_df
"{epithelium}/{robj_dir}/epithelium_nm_df.rds" %>%
glue::glue() %>%
here::here() %>%
saveRDS(
object = nm_df,
file = .)
# new_cn <- data.frame(mod_nm = colnames(decon_mtrx)) %>%
# dplyr::left_join(nm_df, by = "mod_nm") %>%
# # Central.Mem.PASK. fives some trouble because it only changes between + an -
# # negative goes first and distinct solves it automatically
# dplyr::distinct() %>%
# dplyr::pull(plt_nm)
#
# colnames(decon_mtrx) <- new_cn
We are going to add the deconvolution to the Seurat object.
rownames(decon_mtrx) <- rownames(sp_obj@meta.data)
sp_obj@meta.data <- sp_obj@meta.data %>%
tibble::rownames_to_column("barcode") %>%
dplyr::left_join(data.frame(decon_mtrx) %>%
tibble::rownames_to_column("barcode"), by = "barcode") %>%
tibble::column_to_rownames("barcode")
Look at cells topic profile
basis_spotlight <- data.frame(NMF::basis(spotlight_ls[[1]][[1]]))
train_labs <- spotlight_ls[[1]][[2]]
colnames(basis_spotlight) <- unique(stringr::str_wrap(train_labs, width = 30))
basis_spotlight[basis_spotlight < 0.0000001] <- 0
DT::datatable(basis_spotlight, filter = "top")
Look at the location of each cell type in each slice separately
# Iterate over cell types
ct <- colnames(decon_mtrx)
# Seurat::Idents(sp_obj) <- sp_obj@meta.data$gem_id
sp_sub <- sp_obj[, sp_obj@meta.data$gem_id == sample_id]
sp_sub@images <- sp_sub@images[Seurat::Images(sp_sub) == sample_id]
ct_plt_ls <- lapply(ct, function(i) {
tmp_plt <- Seurat::SpatialFeaturePlot(object = sp_sub,
features = i,
alpha = c(0, 1)) +
ggplot2::scale_fill_gradientn(
colors = heat.colors(10, rev = TRUE)) +
ggplot2::scale_alpha(range = c(0, 1)) +
ggplot2::labs(title = stringr::str_wrap(string = i,
width = 25),
fill = "") +
ggplot2::theme(
plot.title = ggplot2::element_text(
hjust = 0.5,
size = 20,
face = "bold"))
if (sum(sp_sub@meta.data[, i]) == 0) {
tmp_plt <- suppressMessages(tmp_plt + ggplot2::scale_alpha(range = c(0,0)))
}
return(tmp_plt)
})
(plt_arr <- cowplot::plot_grid(
plotlist = ct_plt_ls,
axis = "trbl",
align = "hv",
nrow = 5,
ncol = 5))